Antibody Preparations

ABSTRACT

An antibody preparation suitable for intravenous administration in humans includes IgG, IgA and at least 5% IgM antibodies by weight of the total amount of antibodies. The preparation is prepared from human plasma, has specific complement activating activity, and, in an in vitro assay with human serum suitable to determine the ability of the antibody preparation to activate complement unspecifically, the antibody preparation generates substantially no C5a and/or substantially no C3a. The antibody preparation can have medical uses.

This application is a Continuation of, and claims priority under 35U.S.C. § 120 to, U.S. patent application Ser. No. 16/029,781, filed Jul.9, 2018, which is a Divisional of U.S. patent application Ser. No.15/348,121, filed Nov. 10, 2016, which is a Divisional of U.S. patentapplication Ser. No. 14/529,400, filed Oct. 31, 2014, now U.S. Pat. No.9,518,110, issued on Dec. 13, 2016, which is a Divisional of U.S. patentapplication Ser. No. 13/655,686, filed Oct. 19, 2012, now U.S. Pat. No.8,900,806, issued on Dec. 2, 2014, which is a Continuation of, andclaimed priority under 35 U.S.C. § 120 to, International Application No.PCT/EP2011/056487, filed Apr. 21, 2011, and claims priority therethroughunder 35 U.S.C. § 119 to Great Britain Patent Application No. 1006753.6,filed Apr. 22, 2010, the entireties of which are incorporated byreference herein.

FIELD OF THE INVENTION

The present invention relates to an antibody (immunoglobulin)preparation comprising IgM which has specific complement activatingactivity but low unspecific complement activation capacity. The presentinvention also relates to the use of the antibody preparation inmedicine.

BACKGROUND OF THE INVENTION

Immunoglobulin compositions prepared from human plasma and suitable forintravenous administration are known in the art and for several decadeshave played an important role in the treatment of a wide range ofdiseases. Immunoglobulins are used, for example, for the treatment ofinfections in humans and can be assigned to various classes with variousbiochemical and physiological properties. Immunoglobulin G participatesin defending against viral antigens, whereas IgM is predominantly activein antibacterial and antitoxin immune responses.

The immunoglobulin solutions comprise IgG, IgA and IgM in variouspercentages, with different preparations having different treatmentapplications, e.g. preparations with a higher percentage of IgM are usedin the prophylaxis or treatment of bacterial infections.

The immunoglobulin solutions are usually prepared from fractions ofblood plasma or serum, e.g. Cohn fractions. These fractions are thensubjected to a number of purification steps to remove contaminants suchas viruses, denatured proteins, proteases and lipids.

Human plasma for fractionation is collected from thousands of donors andmay contain pathogen viruses despite thorough testing of the sourceplasma. Therefore process steps to inactivate or remove viruses areessential in order to achieve safe products for use in medicine. Severaltechniques for virus inactivation/removal are known in the art, e.g.chemical treatments, irradiation with UVC light or nanometer filtration,which are performed in order to ensure overall virus safety.

The virus removal or inactivation capacity of the process steps isvalidated using laboratory scale models of the production process andfor each step a removal or inactivation factor is determined. Anincrease of the inactivation/removal factor adds additional viral safetyto the pharmaceutical product. Today guidelines from regulatoryauthorities require at least two effective steps for enveloped andnon-enveloped viruses in the manufacture of plasma-derivedpharmaceuticals. Although several methods, such as solvent/detergenttreatment, octanoic acid treatment, nanometer filtration and heattreatment, are effective to inactivate or remove enveloped viruses thereare only a few methods known to inactivate or remove non-envelopedviruses, for example Parvo viruses. These non-enveloped viruses aremostly very small, usually passing through nanometer filters with poresizes above 20 nm. This pore size is too small for IgM molecules havinga diameter up to 30 nm. Non enveloped viruses are effectivelyinactivated by chemicals like ß-propiolactone which, however, also leadsto a modified immunoglobulin with impaired functions. Another effectivetreatment is UVC-irradiation (EP1842561, CAF-DCF). However, knownsolvent/detergent treatments, octanoic acid treatment and mild heattreatment have no substantial effect on non-enveloped viruses.

As mentioned above, in addition to viruses which are potentially presentit is also necessary to remove other contaminants like lipids,proteases, protein aggregates, and denatured immunoglobulins. Theremoval of all these contaminants is essential (1) to ensure the productcomplies with bio-safety guidelines regarding viral contamination, (2)in order for the product to be tolerated by the patient afterintravenous administration, (3) to allow the product to be stable duringlong-term storage (any residual proteolytic activity might lead todegradation of the product over long-term storage, e.g. 2 years), and(4) to generate the desired compound mixture/pharmaceutical composition.

At the same time, however, it is essential that the purification stepsto remove the contaminants do not interfere with the immunoglobulinmolecules, so that as far as possible these retain their normalbiological activity and are retained at high yield in solution. Thisbalance is difficult to achieve since many known purification steps canalso have a negative impact on the activity of the immunoglobulins, andin particular on IgM; for example extended irradiation times with UVCcan reduce the yield of native and active IgM obtained in the finalimmunoglobulin solution. Not only does this lead to a reduction inefficacy of the final immunoglobulin solution but it can also cause thesolution to be less well tolerated in vivo.

Aggregates and denatured immunoglobulins, the amount of which can beincreased by certain purification steps, especially are a potential riskfor the patients because they have a high capacity to activatecomplement unspecifically, leading to severe side effects in patientsreceiving these denatured immunoglobulins. Unspecific complementactivation refers to the initiation of the complement cascade in theabsence of specific antibody-antigen complexes. Unspecific complementactivation is strictly to be avoided since it may cause undesirable sideeffects such as hypotension, flushing, headache, fever, chills, nausea,vomiting, muscle pain, dyspnoea and tachycardia. Specific complementactivation, on the other hand, is desirable and it occurs only after theimmunoglobulins have bound to their specific antigens.

Unspecific complement activation is measured as the so calledanticomplementary activity (ACA) by a standardized test described in theEuropean Pharmacopoeia.

The role of the complement system in the immune defense of pathogens iswell known. The complement system consists of about 20 proteins, whichare activated sequentially. The classical complement pathway typicallyrequires a specific antigen antibody complex for activation, whereas thealternative pathway can be activated by antigens without the presence ofantibodies. The classical and the alternative pathway of complementactivation all generate a protease C3-convertase. The C3-convertasecleaves and activates component C3, creating C3a and C3b, and causing acascade of further cleavage and activation events over C5 convertase toC5a and C5b. C5b initiates the membrane attack pathway, which results inthe membrane attack complex, consisting of C5b, C6, C7, C8, andpolymeric C9. This is the cytolytic endproduct of the complement cascadewhich forms a transmembrane channel, which causes osmotic lysis of thetarget cells like bacteria.

Complement activation additionally results in the formation ofanaphylatoxins, including the biologically active protein C5a. Thisanaphylatoxin is a potent chemotactic agent for immune and inflammatorycells and induces cell activation and causing the release of histaminefrom mast cells. In situations of excessive or uncontrolled and/orunspecific complement activation, the overproduction of C5a can causedeleterious effects to patients.

C5a is an effective leukocyte chemoattractant, causing the accumulationof white blood cells, especially neutrophil granulocytes, at sites ofcomplement activation. C5a activates white blood cells and is a powerfulinflammatory mediator. Whereas these functions are beneficial duringspecific antibody-antigen complex reactions all unspecific generation ofC5a has to be avoided due to the potential side effects.

Unspecific complement activation is a particular issue for IgMimmunoglobulin preparations (i.e. those comprising at least 5% IgM) asin contrast to IgG preparations IgM antibodies easily aggregate insolution. IgM preparations are difficult to stabilize especially if theyare enriched compared to plasma concentrations and stored in liquidsolution. It is also known that IgM is a vigorous activator ofcomplement; a single molecule bound to an antigen can activatecomplement. This is in contrast to IgG, where two or more molecules ofIgG must be bound to an antigen in close association with each other toactivate complement.

Still further, the main indications treated by IgM containingimmunoglobulin preparations are bacterial infections and sepsis. Asthese patients are already suffering from hypotension an additionalunwanted generation of unspecific complement activation and C5a wouldlead to a clinical worsening of the patient's condition. Accordingly,IgM preparations have been described as being difficult to prepare forintravenous application.

There are several methods described in the art for the production of IgMcontaining immunoglobulin preparations from human plasma.

The initial purification of human IgM solutions has been carried out byclassical Cohn plasma fractionation methods or its well knownmodifications (e.g. Cohn/Oncley, Kistler/Nitschmann). Using cold ethanolprecipitation processes the IgM fraction is recovered in fraction III orfraction I/III (also called B or B+I). Starting from fraction III orI/III methods have been described for purification of protein solutionsenriched in IgM EP0013901 describes a purification method starting fromfraction III including steps using octanoic acid, β-Propiolactonetreatment and an adsorption step using an anionic exchange resin. Thismethod is used to produce Pentaglobin®- to date the only commerciallyavailable intravenous IgM product. β-propiolactone is a well knownchemical used in sterilization steps in order to inactivate viruseswhich are potentially present. As β-propiolactone is a very reactivesubstance which causes the chemical modification of proteins there isalso substantial loss of the anti-viral and anti-bacterial activities ofthe immunoglobulins. On the other hand this chemical modificationresults in an reduced anticomplementary activity compared to anchemically unmodified immunoglobulin. EP0352500 describes thepreparation of an IgM concentrate for intravenous application with areduced anti-complementary activity by using anionic exchangechromatography, β-Propiolactone, UVC light irradiation and an incubationstep at increased temperature (40° C. to 60° C.). The preparationproduced by this method was stable in liquid solution for a limited timedue to the chemical modification. The IgM concentration was above 50%from the total immunoglobulin content.

The preparation of protein solutions enriched in IgM without chemicalmodification by β-propiolactone has been described in EP0413187(Biotest) and EP0413188 (Biotest). These methods involve subjecting asuitable protein solution to octanoic acid treatment and anionicexchange chromatography, starting from Cohn fraction III or II/III. Inpatent EP0413187 (Biotest) the octanoic acid treatment is carried out bystirring for 15 min, in order to remove lipids being present in Cohnfraction III.

The preparation according to EP0413187 had a low anticomplementaryactivity, between 0.6 and 0.8 CH50/mg protein, but had to be stabilizedand virus inactivated by ß-propiolactone. Low anticomplementary activityis considered to be ≤1 CH50/mg protein according to EP monograph forimmunoglobulins.

EP0413188B1 (Biotest) describes the preparation of an IgM-enrichedpreparation for intravenous administration by using an anion exchangechromatography in order to reduce the anti-complementary activity.Additionally a heat treatment at pH 4-4.5 at 40 to 60° C., preferablybetween 50 and 54° C., was described to reduce the anticomplementaryactivity. This preparation had to be lyophilized to ensure stability ofthe preparation for several months. Long term stability as a liquidsolution could not be shown.

M. Wickerhauser et al. “Large Scale Preparation of Macroglobulin”, VoxSang 23, 119-125 (1972) showed that IgM preparations isolated by PEGprecipitation had high anticomplementary activity (ACA) by a standardcomplement fixation test and this ACA activity was reduced 10 fold byincubating the IgM preparation at pH 4.0 at 37° C. for 8 hours followedby readjustment to neutral pH. It was not demonstrated if this 10 foldreduction is sufficient to ensure intranenous tolerability. The authorsdid not assess the specific complement activating potential of their IgMconcentrate, nor did they assess safety in any animal or human model.

Another method describes the use of mild-heat treatment of IgMpreparations at 40 to 62° C., preferably 45 to 55° C., at pH 4.0 to 5.0(EP 0450412, Miles) to reduce the unspecific complement activation. Inthis patent application octanoic acid is added to a Cohn fraction IIIsuspension in order to remove prekallikrein activator and lipoproteinsby centrifugation. Nevertheless this mild heat treatment led to partialloss of antigenic determinants of IgM. This may increase the risk ofgenerating neo-antigens leading to a increased immunogenicity in humansor the loss of activity.

The preparation of an IgM containing protein solution for intravenousapplication by using a protease treatment (e.g. with pepsin) after anoctanoic acid precipitation step has been described in EP0835880 (U.S.Pat. No. 6,136,312, ZLB). Protease treatment leads to partialfragmentation of the immunoglobulin molecule impairing the fullfunctional activity of the Fab and Fc parts. Therefore protease-treatedimmunoglobulins cannot be regarded as unmodified. Also this preparationmethod leads to about 5% fragments with a molecular weight of <100 kD.

The described methods to carry out the octanoic acid treatment(EP0413187 and EP0835880) have the drawback that the octanoic acidtreatment is not effective with respect to removal and inactivation ofnon-enveloped viruses, and does not remove substantially all proteolyticactivity.

In EP 0345543 (Bayer, Miles) a highly concentrated IgM preparation withat least 33% IgM for therapeutic use is disclosed, the preparation beingsubstantially free of isoagglutinin titres. In this patent applicationan octanoic acid precipitation is carried out by adding the octanoicacid and the isoagglutinins are removed by Synsorb affinitychromatography. The final preparation had to be freeze dried.

Altogether the production of an IgM containing preparation with lowanticomplementary activity is possible if the immunoglobulins arechemically or enzymatically modified and/or further purified bychromatography and/or subjected to a mild heat treatment. However, thesemethods have their drawbacks in the lack of virus removal/virusinactivation (and therefore virus safety), reduction in the amount ofimmunoglobulin molecules in native form and/or residualanticomplementary activity. As such, there is still a need to provideimproved IgM containing immunoglobulin preparations suitable forintravenous administration in humans.

SUMMARY

In a first aspect the subject matter of this application provides IgG,IgA and at least 5% IgM antibodies by weight of the total amount ofantibodies, wherein the preparation is prepared from human plasma,wherein the antibody preparation has specific complement activatingactivity and wherein in an in vitro assay with human serum suitable todetermine the ability of the antibody preparation to activate complementunspecifically the antibody preparation generates substantially no C5aand/or substantially no C3a.

It is a further aspect of the subject matter of this application toprovide an antibody preparation as described above, further comprisingmore than 5% IgA and more than 40% IgG, and/or at least 10%, or 15%, IgMby weight of the total amount of antibodies.

It is a further aspect of the subject matter of this application toprovide the antibody preparation as described above, wherein theantibody preparation adjusted to an IgM concentration of 1.72 mg/mlgenerates less than 200 ng/ml C5a after 60 minutes of the assay.

It is a further aspect of the subject matter of this application toprovide the antibody preparation as described above, An antibodypreparation according to any preceding claim wherein the antibodypreparation adjusted to an IgM concentration of 1.72 mg/ml generatesless than 6000 ng/ml C3a after 60 minutes of the assay.

It is a further aspect of the subject matter of this application toprovide the antibody preparation as described above, wherein in the invitro serum assay the antibody preparation with human serum generatesthe same amount of C5a and/or C3a as human serum alone ±70% to determinethat the antibody preparation generates substantially no C5a comprisesthe steps of: (a) adding an amount of the antibody preparation to 100 μlhuman serum to create a reaction mixture containing 1.72 mg/ml IgM andincubating the reaction mixture for 60 minutes at 37° C. with constantagitation; (b) preparing a set of dilutions of the reaction mixturesuitable for an ELISA; (c) performing a sandwich ELISA on the set ofdilutions of the reaction mixture utilizing a primary and a secondaryantibody to C5a and a chromogenic substance, wherein the secondaryantibody is conjugated to an enzyme and the chromogenic substance is thesubstrate of the enzyme; and (d) determining the amount of C5a in thereaction mixture based on a colour change obtained as a result ofcontacting the chromogenic substance with the enzyme bound to C5a viathe secondary antibody.

It is a further aspect of the subject matter of this application toprovide the antibody preparation as described above, wherein the invitro serum assay to determine that the antibody preparation generatessubstantially no C3a comprises the steps of: (a) adding an amount of theantibody preparation to 100 μl human serum to create a reaction mixturecontaining 1.72 mg/ml IgM and incubating the reaction mixture for 60minutes at 37° C. with constant agitation; (b) preparing a set ofdilutions of the reaction mixture suitable for an ELISA; (c) performinga sandwich ELISA on the set of dilutions of the reaction mixtureutilizing a primary and a secondary antibody to C3a and a chromogenicsubstance, wherein the secondary antibody is conjugated to an enzyme andthe chromogenic substance is the substrate of the enzyme; and (d)determining the amount of C3a in the reaction mixture based on a colourchange obtained as a result of contacting the chromogenic substance withthe enzyme bound to C3a via the secondary antibody.

It is a further aspect of the subject matter of this application toprovide the antibody preparation as described above, comprising lessthan 2% or 1.5% aggregates of 1200 kDa or above.

It is a further aspect of the subject matter of this application toprovide the antibody preparation as described above, wherein theanti-complementary activity of the preparation is less than 1.0 or 0.75CH50/mg protein.

It is a further aspect of the subject matter of this application toprovide the antibody preparation as described above, comprising animmunoglobulin content of greater than 95% of the total protein content.

It is a further aspect of the subject matter of this application toprovide the antibody preparation as described above, prepared from humanserum in the absence of a step of heat treatment at a temperature 40° C.or above for more than 10 minutes.

It is a further aspect of the subject matter of this application toprovide the antibody preparation as described above, prepared from humanserum in the absence of a step involving chemical or enzymaticmodification of the antibodies.

It is a further aspect of the subject matter of this application toprovide the antibody preparation as described above, wherein the step ofchemical modification is a step of contacting the antibodies withβ-propiolactone.

It is a further aspect of the subject matter of this application toprovide the antibody preparation as described above, prepared by aprocess which is capable of a more than 31 ogio removal of non-envelopedviruses.

It is a further aspect of the subject matter of this application toprovide the antibody preparation as described above, prepared from humanplasma by a process comprising the steps of: (a) preparing from thehuman plasma a plasma fraction as a solution containing immunoglobulins;(b) mixing a C₇ to C9 carboxylic acid with the solution and treating themixed solution with a vibrating agitator to precipitate contaminatingproteins; (c) separating the precipitated proteins from the solution toyield the IgM containing immunoglobulin composition; (d) incubating theIgM containing immunoglobulin composition at between pH3.5 and pH 4.5 toform an incubated solution; (e) irradiating the incubated solution withUVC to form a UVC irradiated solution; and (f) filtering the UVCirradiated solution under sterile conditions to form the antibodypreparation suitable for intravenous administration in humans.

It is a further aspect of the subject matter of this application toprovide the antibody preparation as described above, wherein the processfurther comprises subjecting the incubated solution obtained from step(d) to nanofiltration prior irradiation in step (e).

It is a further aspect of the subject matter of this application toprovide the antibody preparation as described above, wherein theantibody preparation is capable of administration to cynomolgus monkeysat 1 15 mg IgM/kgBW/hr in the absence of a greater than 10% reduction inarterial pressure from pretreatment level.

It is a further aspect of the subject matter of this application toprovide the antibody preparation as described above, wherein at least90% of the antibodies in the preparation are biologically active.

It is a further aspect of the subject matter of this application toprovide the antibody preparation as described above, wherein in an invitro Rubella antigen based assay suitable to determine Fc function theactivity of the Fc part of the antibodies of the antibody preparation isthe same as that of a biological reference preparation ±10%.

It is a further aspect of the subject matter of this application toprovide a method of producing an antibody preparation according to anypreceding claim from human plasma comprising the steps of: (a) preparingfrom the human plasma a plasma fraction as a solution containingimmunoglobulins; (b) mixing a C₇ to C9 carboxylic acid with the solutionand treating the mixed solution with a vibrating agitator to precipitatecontaminating proteins; (c) separating the precipitated proteins fromthe solution to yield the IgM containing immunoglobulin composition; (d)incubating the IgM containing immunoglobulin composition at betweenpH3.5 and pH 4.5 to form an incubated solution; (e) irradiating theincubated solution with UVC to form a UVC irradiated solution; and (f)filtering the UVC irradiated solution under sterile conditions to formthe antibody preparation suitable for intravenous administration inhumans.

It is a further aspect of the subject matter of this application toprovide an antibody preparation as described above for use in medicine,for example, in the treatment of an immunological disorders or abacterial infection.

It is a further aspect of the subject matter of this application toprovide an antibody preparation as described above, wherein theimmunological disorder is an IgM deficiency disorder.

It is a further aspect of the subject matter of this application toprovide a method of using an antibody preparation as described above forthe manufacture of a medicament for treatment of an immunologicaldisorder or a bacterial infections.

It is a further aspect of the subject matter of this application toprovide a method of treatment of a patient comprising administering anantibody preparation as described above.

It is a further aspect of the subject matter of this application toprovide a method of treatment as described above, wherein the patient issuffering from an immunological disorder or a bacterial infection.

It is further aspect of the subject matter of this application toprovide a method of treatment as described above, wherein the antibodypreparation is administered to the patient intravenously

The present applicants have surprisingly found that the production of anIgM antibody preparation from human serum is possible which has specificcomplement activating activity and substantially no unspecificcomplement activity. This product is advantageous as it maintainsproduct efficacy while reducing unwanted side-effects such ashypotension, associated with unspecific complement activation afterintravenous administration.

A further aspect of the subject matter of this application provides amethod of producing the antibody preparation of the present inventionfrom human plasma comprising the steps of:

(a) preparing from the human plasma a plasma fraction as a solutioncontaining immunoglobulins;

(b) mixing a C₇ to C₉ carboxylic acid with the solution and treating themixed solution with a vibrating agitator to precipitate contaminatingproteins;

(c) separating the precipitated proteins from the solution to yield theIgM containing immunoglobulin composition;

(d) incubating the IgM containing immunoglobulin composition at betweenpH3.5 and pH 4.5 to form an incubated solution;

(e) irradiating the incubated solution with UVC to form a UVC irradiatedsolution; and

(f) filtering the UVC irradiated solution under sterile conditions toform the antibody preparation suitable for intravenous administration inhumans.

The present applicants have surprisingly found that the use of avibrating agitator during the step where the immunoglobulin solution ismixed with the carboxylic acid is extremely advantageous. This methodstep provides a more efficient removal of unwanted proteins (includingproteases) and produces an intermediate product which is better suitedto downstream processing steps utilised to produce an immunoglobulinmedicament; the intermediate product allows these downstream processingsteps to be more efficient. Accordingly, the downstream processing stepscan be less harsh, helping to achieve the antibody preparation of thepresent invention which is capable of specific complement activation andsubstantially no unspecific complement activation.

In particular, the IgM immunoglobulin containing composition obtainedfrom step (c) can be combined with further treatment steps, such astreatment with mild acid conditions and treatment with UVC irradiation,to produce an IgM containing immunoglobulin product or antibodypreparation which is suitable for intravenous administration and whichhas the following advantageous properties: having low anti-complementaryactivity; retaining a high level of native and active IgM; and beingvirus safe and thus suitable for intravenous administration in humans.The level of virus safety achieved with the methods described herein hasnot previously been obtaininable. Additional advantages are having lowproteolytic activity (and therefore being stable during long termstorage) and being chemically unmodified.

Still further, the subject matter of this application provides anantibody preparation for use in medicine. In one embodiment the antibodypreparation is for use in the treatment of immunological disorders andbacterial infections.

A further aspect of the subject matter of this application provides amethod of treatment comprising administering the antibody preparation ofthe present invention to a patient.

The present invention will now be described in further detail by way ofexample only, with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides an overview of the steps that can be utilised to form anantibody preparation suitable for intravenous administration accordingto the present invention. The octanoic acid treatment step employing avibromixer device, the pH4 treatment and the UVC treatment arehighlighted The starting material is generated from a standard coldethanol precipitation process of human plasma.

FIG. 2 provides a graph showing time dependent mean C5a concentrationsfound in human serum after incubation with IgM preparations.

FIG. 3 provides a graph showing time dependent mean C3a concentrationsfound in human serum after incubation with IgM preparations.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Antibody Preparation

As described above, the present invention provides an antibodypreparation suitable for intravenous administration in humans comprisingIgG, IgA and at least 5% IgM antibodies by weight of the total amount ofantibodies, wherein the preparation is prepared from human plasma,wherein the antibody preparation has specific complement activatingactivity, and wherein in an in vitro assay with human serum suitable todetermine the ability of the antibody preparation to activate complementunspecifically the antibody preparation generates substantially no C5aand/or substantially no C3a.

The antibody preparation of the present invention comprises human plasmaproteins of which at least 90%, preferably at least 95% is made up ofimmunoglobulins (polyclonal antibodies). In particular the preparationcomprises the immunoglobulins IgG, IgA and IgM wherein at least 5% ofthe immunoglobulins are IgM. The amount of IgG, IgA and IgMimmunoglobulins can be determined by nephelometry or byimmunoprecipitation according to Ph. Eur. 2.7.1.

More preferably the antibody preparation comprises at least 10% IgM andmost preferably at least 15% IgM. In relation to IgG and IgA, preferablythe antibody preparation comprises more than 5% IgA and/or more than 40%IgG. All percentages are percentage of total amount of antibodies (forexample, g of IgM/(g of IgG+g of IgA+g of IgM)×100).

Methods for determining that the antibody preparation has specificcomplement activating activity (i.e. the ability to activate thecomplement cascade in the presence of antigen) through assessing thefunctional activity of the Fc part of the immunoglobulin molecule areknown in the art. In particular, a suitable method is described by thecurrent Eur. Ph. method according to the European Guidelines ICH S6(CPMP/ICH/302/95) which utilizes Rubella antigen. Further detailsregarding specific complement activation are provided below in referenceto biological activity.

The antibody preparation causes substantially no unspecific complementactivation (i.e. activation of the complement cascade by immunoglobulinsin the absence of antigen) in in vitro assays suitable to determineunspecific complement activation in normal human serum (i.e. serum fromhealthy humans). In particular, the assay can determine the amount ofC5a and/or C3a generated in the assay in the absence of antigen. Asnoted above, complement activation results in the production of C5a andC3a. Since both of these proteins are involved in the terminal pathwayof the complement system (rather than in either the classical/lectinpathway or the alternative pathway) they are particularly useful todetermine complement activation.

The antibody preparation generates substantially no C5a and/orsubstantially no C3a when used in an appropriate in vitro assay withhuman serum in the absence of antigen. In a preferred embodiment theantibody preparation adjusted to an IgM concentration of 1.72 mg/mlgenerates less than 200 ng/ml C5a after 60 minutes of the assay, and/orthe antibody preparation adjusted to an IgM concentration of 1.72 mg/mlgenerates less than 6000 ng/ml C3a after 60 minutes of the assay.

Alternatively, or in addition, the amount of C5a and/or C3a generated bythe antibody preparation in the assay is the same as the amount of C5aand/or C3a generated in the same assay by human serum alone ±70%.Preferably this is after 60 minutes of the assay.

Suitable assays are known in the art. In a preferred embodiment theassay comprises the steps of:

(a) adding an amount of the antibody preparation to 100 μl human serumto create a reaction mixture containing 1.72 mg/ml IgM and incubatingthe reaction mixture for 60 minutes at 37° C. with constant agitation;(b) preparing a set of dilutions of the reaction mixture suitable for anELISA;(c) performing a sandwich ELISA on the set of dilutions of the reactionmixture utilizing a primary and a secondary antibody to C5a or C3a and achromogenic substance, wherein the secondary antibody is conjugated toan enzyme and the chromogenic substance is the substrate of the enzyme;and(d) determining the amount of C5a or C3a in the reaction mixture basedon a colour change obtained as a result of contacting the chromogenicsubstance with the enzyme bound to C5a or C3a via the secondaryantibody.

In the ELISA the set of dilutions are contacted with wells of an assayplate coated with the primary antibody. After incubation the wells arewashed to remove the dilution sample, The second antibody is thenincubated and binds to any C3a/C5a bound to the primary antibody in thewells, since it has a different epitope on the C3a/C5a to the primaryantibody. After further washing to remove unbound secondary antibody thechromogen is incubated and reacts with the enzyme conjugated to thesecond antibody. The resulting colour change can be measured via opticaldensity determinations with a photometer, being proportional to theconcentration of C5a/C3a present in the set of dilutions.

In particular, the amount of the antibody preparation added in step (a)is that appropriate to create a concentration of 1.72 mg/ml IgM in thereaction mixture. Steps (c) and (d) can comprise: (i) applying the setof dilutions of the reaction mixture to the wells of an assay platewhich are coated with a primary antibody to C3a/C5a (i.e. “the captureantibody”); (ii) incubating the plate to allow any C3a/C5a to bind theprimary antibody; (iii) washing the plate to remove any material in thedilutions not bound to the primary antibody; (iv) applying a secondaryenzyme linked antibody (the detection antibody) that also binds toC3a/C5a; (v) incubating the plate to allow any secondary antibody tobind to C3a/C5a; (vi) washing the plate to remove unbound secondaryantibody; (vii) applying a chemical that is converted by the enzyme intoa colour signal; and (viii) measuring the absorbency of the plate wellsto determine the presence and quantity of C3a/C5a.

The sandwich ELISA is performed according to methods known in the art,and/or with commercially available kits according to manufacturer'sinstructions. Suitable, and particularly preferred, commerciallyavailable enzyme linked immunosorbent assay (ELISA) kits are QuidelMicroVue C5a Plus EIA Kit; A025, and Quidel MicroVue C3a Plus EIA Kit;A032.

In a further embodiment of the present invention the antibodypreparation comprises less than 2% aggregates of 1200 kDa or above,preferably less than 1.5%. This refers to the % of the immunoglobulincontent. The amount of aggregates can be determined by high performancesize exclusion chromatography (HPSEC). This can be performed by methodsknown in the art.

Alternatively, or in addition the ability of the antibody preparation togenerate substantially no unspecific complement activation can bedefined as the anti-complementary activity of the preparation being lessthan 1.0 CH50/mg protein, more preferably less than 0.75 CH50/mgprotein. The assay to determine the anti-complementary activity on thisscale can be carried out according to the method described in theEuropean Pharmacopoeia (method 2.6.17, Ph. Eur. 6. Edition, 2008).Further details of this assay are provided in the assay section below.

In a preferred embodiment the antibody preparation has been preparedfrom human serum in the absence of a step involving chemical orenzymatic modification of the antibodies, i.e. the process of productionof the antibody preparation from human serum does not comprise a step ofcontacting the antibodies with a reagent which would cause theirenzymatic or chemical modification. In particular, the process does notcomprise contacting the antibodies with β-propiolactone, which causeschemical modification of the antibodies, or comprise contacting theantibodies with pepsin, which would cause enzymatic cleavage of theantibodies.

Alternatively, or in addition, the antibody preparation has beenprepared from human serum in the absence of a step involving heating ofthe antibodies to a temperature of 40° C. or more for 10 minutes ormore. In particular, it is known that heating steps can denature theimmunoglobulins and causes immunoglobulin aggregation.

Further preferably the antibody preparation is prepared by a processwhich is capable of a more than 3 log₁₀, preferably more than 4 log₁₀,and most preferably by more than 5 log₁₀. removal of non-envelopedviruses, thus making the antibody preparation virus safe. The antibodypreparation is therefore safer than the antibody preparations of theprior art, particularly with respect to active non enveloped viruseslike, for example, parvoviruses. This results in an antibody preparationthat is substantially free of virus, and in particular substantiallyfree of non-enveloped virus. Still further, the method of the presentinvention is able to achieve this level of viral particleremoval/inactivation without a significant impact on the amount ofactive IgM or on the anticomplementary activity of the antibodypreparation.

In particular, the antibody preparation can be prepared from humanplasma by a process comprising the steps of:

(a) preparing from the human plasma a plasma fraction as a solutioncontaining immunoglobulins;

(b) mixing a C₇ to C₉ carboxylic acid with the solution and treating themixed solution with a vibrating agitator to precipitate contaminatingproteins;

(c) separating the precipitated proteins from the solution to yield theIgM containing immunoglobulin composition;

(d) incubating the IgM containing immunoglobulin composition at betweenpH 3.5 and pH 4.5 to form an incubated solution;

(e) irradiating the incubated solution with UVC to form a UVC irradiatedsolution; and

(f) filtering the UVC irradiated solution under sterile conditions toform the antibody preparation suitable for intravenous administration inhumans.

It is preferred that the process further comprises subjecting theincubated solution obtained from step (d) to nanofiltration priorirradiation in step (e). Further details and preferred aspects of themethod of production are described in the section below.

In a further preferred embodiment of the invention the antibodypreparation is capable of administration to cynomolgus monkeys at 115 mgIgM/kg body weight/hour in the absence of a 10% or greater reduction inarterial pressure from pretreatment level. As noted above, unspecificcomplement activation causes hypotension and therefore the lack of asignificant change in arterial pressure indicates that substantially nounspecific activation of complement is occurring in healthy monkeys invivo. Arterial pressure can be measured by inserting a pressure catheterinto the lower abdominal aorta via the right fermoral artery.

In a preferred embodiment the antibody preparation also comprisesantibodies against one or more of Pneumococcus saccharide, Escherichiacoli, Enterococcus faecalis, Candida albicans, and Chlamydia.

In a further preferred embodiment at least 90% of the antibodies in theantibody preparation are biologically active. The term biologicallyactive means that the antibodies in the preparation are in native formand in particular are capable of activation of the complement cascade asa result of specific binding to an antigen. The biological activity ofan antibody preparation can be assessed based on assays to determineantibody titre/binding activity and Fc integrity/function known in theart. In particular, in an in vitro Rubella antigen based assay suitableto determine Fc function the activity of the Fc part of the antibodiesof the antibody preparation is the same as that of a biologicalreference preparation ±10%, more preferably ±5%.

Biological reference preparations are utilised by the internationalmedical and healthcare community and help to ensure consistency inmedical products. As such, suitable biological reference preparationsfor the assay are known and are available in the art (e.g.Immunoglobulin Biological Reference Preparation (Batch No. 3). Inparticular, the assay can be performed according to the Internationallyrecognised Eur. Ph. 2.7.9 Test for Fc Function of Immunoglobulin(current edition April 2011) which utilises Human ImmunoglobulinBiological Reference Preparation (Batch No. 3) as a control, againstwhich the % activity of the antibody preparation is determined. Thistest comprises the steps of (i) loading tanned group O human red bloodcells with rubella virus antigen to create antigen coated blood cells;(ii) incubating an amount of the antibody preparation with the bloodcells; adding guinea pig complement to start complement initiated lysesof blood cells; (iii) measuring the kinetic of haemolysis viatime-dependent changes of absorbance at 541 nm; (iv) evaluating thefunction of the antibodies of the antibody preparation using the maximalchange of absorbance per time.

The antibody preparation preferably also has a lower proteolyticactivity than the antibody preparations described in the prior art. Inparticular, no proteolytic activity is detectable in the preparationwhen it is stored at between 2 to 8° C. The proteolytic activity can bemeasured by standardized test methods known in the art, such as thatusing the chromogenic substrate which is described in the assay sectionbelow, and in Example 6.

The antibody preparation of the present invention may further comprise astabilizing agent, such as glycine.

As with preparations known in the art the antibody preparation of thepresent invention can be stored at 5±3° C. However, due to the efficientpurification with the method of the present invention the stability ofthe antibody preparation is extremely good. The final product is stablein liquid form for at least 3 months, preferably at least 6 months andmost preferably at least two years at 2 to 8° C., which means that thereis no fragmentation or polymerization of IgM above 1.5% measured inHPSEC, no increase of proteolytic activity, no decrease of IgM antibodyactivity against Escherichia coli and IgM antibody activity againstPneumococcus saccharide of more than 25% and no increase inanticomplementary activity of more than 25%, staying below 1 CH50/mgprotein. Still further, the final product produced by the method of thepresent invention is stable in liquid form for at least 3 months,preferably at least 6 months, and most preferably at least one year atroom temperature (between 23 and 27° C.) as assessed by the samecriteria.

Method of Production of Antibody Preparation

As described above, the present invention provides a preparation of anIgM containing antibody preparation from a plasma fraction comprisingimmunoglobulins. In particular, the present invention provides a methodof producing the antibody preparation described herein from human plasmacomprising the steps of:

(a) preparing from the human plasma a plasma fraction as a solutioncontaining immunoglobulins;

(b) mixing a C₇ to C₉ carboxylic acid with the solution and treating themixed solution with a vibrating agitator to precipitate contaminatingproteins;

(c) separating the precipitated proteins from the solution to yield theIgM containing immunoglobulin composition;

(d) incubating the IgM containing immunoglobulin composition at betweenpH3.5 and pH 4.5 to form an incubated solution;

(e) irradiating the incubated solution with UVC to form a UVC irradiatedsolution; and

(f) filtering the UVC irradiated solution under sterile conditions toform the antibody preparation suitable for intravenous administration inhumans.

Plasma fractions suitable for the preparation of pharmaceuticalimmunoglobulin compositions, and methods for their production are wellknown in the art. The plasma fraction is preferably a precipitatedplasma fraction and most preferably a precipitated plasma fractionobtained by the process of Cohn fractionation or its well knownmodifications (e.g. Kistler-Nitschmann). Most preferably the fraction isfraction I/III or fraction III (also known as fraction B+I or fractionB) out of cold ethanol fractionation. It is preferred that theimmunoglobulins of the plasma fraction comprise at least 5% IgM.

Step (a) comprises providing a plasma fraction as a solution containingthe immunoglobulins. In many cases the plasma fraction containing theimmunoglobulins will be in solid or semi-solid form. Thus the aim ofthis step is to ensure or to bring the protein of the plasma fractioninto solution such that it is in a suitable state for mixing with thecarboxylic acid in step (b). This step may comprise mixing the plasmafraction with a suitable buffer. Preferably the buffer is of lowmolarity (i.e. less than 1M) and has a pH between 4.5 and 5.5 e.g. 0.1 Msodium acetate buffer pH 5.05±0.1. Mixing can be completed using a blademixer or a vibrating agitator.

In step (b) the solution formed in step (a) is mixed using a vibratingagitator with a C₇ to C₉ carboxylic acid to precipitate contaminatingproteins (e.g. proteases, viruses etc). The carboxylic acid may bebranched and/or may include substituents which do not substantiallyalter the effect of step (b). The carboxylic acid is preferably octanoicacid. The carboxylic acid is preferably added at a concentration of atleast 0.075 kg/kg of plasma fraction, up to a concentration of 0.2kg/kg. More preferably the carboxylic acid is added at 0.8 to 0.15 kg/kgof plasma fraction, and most preferably between 0.09 kg/kg and 0.13kg/kg. Acid of any convenient molarity may be used to provide thecorrect concentration.

Any type of commercially available vibrating agitator, suitable for usein the chemical/pharmaceutical industry, may be used. Examples ofsuitable vibrating agitators are available from Graber+Pfenninger GmbH.In particular, the “Labormodell Typ 1” vibromixer can be used for labscale experiments, and the “Industriemixer Typ 4” can be used forproduction scale preparations. The vibrating mixers can be usedaccording to manufacturer's instructions, and in particular at settingswhich are described by the manufacturers as suitable for mixingsolutions containing proteins. For example the vibrating mixers canusually be operated at less than 100 Hz with an amplitude less than 10mm, e.g. the vibration mixing using the “Labormodell Typ 1” at lab scalewas carried out by the present inventors at 50 Hz, when 230 V powersupply is used. The vibration amplitude of the mixing process was variedbetween 0 and 3 mm, and for the IgM preparation preferably 3 mm wasused. Stirrer plates with a diameter between 23 mm and 65 mm were usedfor lab scale experiments. For production scale a stirrer plate diameterof 395 mm was used (hole diameters of 13.5 mm and 16 mm).

In step (b) the pH of the mixed solution is preferably between 4.5 to5.5, and more preferably between pH 4.8 and pH 5.3. The step can becarried out in sodium acetate buffer, and, for example, withapproximately 0.1 M sodium acetate buffer. The temperature at which step(b) is conducted is preferably between 10° C. and 35° C., and morepreferably 14 to 30° C.

The mixing time using the vibrating agitator is not particularly limitedbut is preferably at least 30 minutes and not more than 3 hours, andmore preferably from 40-110 minutes. Incubation times of less than 30minutes can reduce the level of virus inactivation.

In one embodiment of step (b) tri-calcium phosphate is mixed with thesolution in step (b). Preferably this is added at 0.01 to 0.02 kg/kgplasma fraction (as it is in solid or semi-solid form). The tri-calciumphosphate can be added simultaneously, separately or sequentially to thecarboxylic acid. In a preferred embodiment the tri-calcium phosphate isadded at least 20 minutes after the carboxylic acid.

In step (c) the contaminating proteins precipitated in step (b) areseparated off from the solution to yield the IgM containingimmunoglobulin composition (i.e. an immunoglobulin containing solution).This step of separation is not particularly limited by can be performedby any suitable method known in the art. However, the separating step ispreferably performed using filtration, and more preferablyultrafiltration, and the result of step (c) is therefore a filteredsolution.

As described above, the method of the present invention is advantageousin manufacturing terms since it appears to cause a more efficientprecipitation of contaminating proteins, and, as a result, step (c) iseasier to perform. When the mixture resulting from step (b) isseparated, a transparently clear solution, i.e. the IgM containingimmunoglobulin composition, is achieved. Filtration is is thereforequicker and easier.

Further process steps (d) to (f) are required to convert the IgMcontaining immunoglobulin composition obtained from step (c) into anantibody preparation suitable for intravenous administration.

Step (d) comprises treating the IgM containing immunoglobulincomposition obtained from step (c) with mild acid conditions, step (e)comprises subjecting the acid treated composition to UVC irradiation toform a UVC irradiated solution, and step (f) comprises filtering the UVCirradiated solution under sterile conditions to form the antibodypreparation suitable for intravenous administration in humans.

For the treatment with mild acid conditions the IgM containingimmunoglobulin composition obtained from step (c) is incubated atbetween pH 3.5 to pH 4.5, and preferably between pH 3.8 and pH 4.2, toform an incubated solution. The mild acid conditions can be created byadding a suitable acid to the IgM containing immunoglobulin composition,for example the pH can be adjusted by adding 0.2M HCl.

This incubation step is preferably carried out at between 32 and 42° C.,and more preferably at between 35 and 39° C. The incubation time ispreferably at least 2 hours and not greater than 24 hours, and morepreferably at least 9 hours but not greater than 16 hours.

In the irradiation step the incubated solution obtained from the mildacid treatment described above is treated with UVC light to form a UVCirradiated solution. This step can be performed using devices which arecommercially available, such as the UVivatec® device (Bayer TechnologyServices). It is preferred that the incubated solution is treated at254±10 nm between 200 and 500 J/m², more particularly between 200 and300 J/m², in order to further inactivate viruses and proteases which arepotentially present. It is noted that UVC treatment under gentleconditions than would normally be required is only possible with thewater-clear filtrate which is obtained by the present invention afterthe octanoic acid treatment with vibromixing. More opalescent or opaquesolutions normally received by standard stirring techniques wouldnecessitate longer irradiation times which would lead to moredenaturation of the IgM activity and lower virus inactivation rates.

In step (f) the irradiated solution is filtering under sterileconditions to form the antibody preparation suitable for intravenousadministration in humans. Preferably the filtration is nanofiltration,more preferably through a filter having a 40 to 50 nm pore size.

In addition to the mild acid treatment the UVC irradiation and thefiltration step, additional steps to achieve an immunoglobulinpreparation for intravenous administration can optionally also compriseone or more further filtration steps. In one embodiment the proteinsolution being processed can be adsorbed onto DEAE-Sephadex and thenseparated from the Sephadex by depth filtration. For example, it mayfurther be subjected to a batch adsorption with 75 mg per kg proteinDEAE Sephadex at pH 5.8, in order to remove the unwanted accompanyingprotein Ceruloplasmin.

In a particularly preferred embodiment the incubated solution obtainedfrom the mild acid treatment is subjected to adsorption ontoDEAE-Sephadex and then separated from the Sephadex by depth filtration,before being treated to UVC irradiation.

In another embodiment the immunoglobulin solution being processed may befiltered through a nanometer filter. Filters of 75±5 nm to 35±5 nm poresize, or filters having a nominal pore size of 75 to 35 nm (for examplePall Ultipor DV50), can be used at various stages during the process. (Anominal pore size of e.g. 50 nm means retention rate of ≥4 log 10 forvirus with size of 50 nm or larger). In a preferred embodiment thesolution obtained from the DEAE-Sephadex step described in the aboveparagraph is filtered through a 0.2 μm filter prior to UVC irradiation.

The final antibody preparation (i.e. the processed IgM containingimmunoglobulin solution) obtained from the process defined above may bedirectly filled into a container under sterile conditions.Alternatively, the antibody preparation may be formulated in aglycine-containing buffer at a pH between 4 and 5.5, and preferablybetween 4.1 to 4.5. The antibody preparation may also be diluted to aprotein concentration between 40 and 80 g/L and preferably between 55and 70 g/L. It is noted that it is also possible to enrich the IgMcontent of the antibody preparation by well known methods like e.g.anion exchange chromatography.

As previously indicated above, the method described above leads to ahigher inactivation and removal of virus particles, especially veryresistant, non-enveloped viruses such as Parvo viruses, which areusually not very susceptible to octanoic acid treatment. Furthermore, animproved removal of proteolytic activity is achieved in comparison toconventional stirring. These features are achieved while keeping a highyield of IgM that is chemically unmodified. This finding contrasts withthe conventional view that the treatment with octanoic acid is not aneffective step against non-enveloped viruses and improved viral safetymust be achieved through inactivation of virus through harsher methodssuch as β-Propiolactone treatment. Also it was well known thatincreasing e.g. octanoic acid concentration to completely removeproteolytic activity results in a massive loss of IgM.

The results of the method are achieved through the use of mixing devicesusing a vibrating mode in combination with the octanoic acid treatment.This is particularly surprising since it is known that IgM is verysusceptible to shear stress, which may lead to an undesired highanticomplementary activity. Accordingly, one would not consider using avibrating mixer to prepare an IgM composition and would not expect sucha favorable impact when using a vibrating mixing during processing of anIgM containing solution.

Furthermore with the method the separation achieved by step (c), such asclarification by filtration of the octanoic acid treated solutionresulting from step (b), is enhanced when a vibrating mixing device isused. Separation is more easily achieved, reducing processing time andmanufacturing costs, and step (c) leads to a limpid solution whichcreates advantages for downstream processing. Conventional solutions,achieved by filtering the results of octanoic acid treated IgMcontaining solutions which have been stirred, are opalescent or opaque.

The resulting IgM containing composition obtained from step (c) ispreferably subjected to treatment with mild acid conditions (e.g. pH 4)and an UVC-irradiation step to further improve virus safety andstabilize the final product. Due to the enhanced clarification of theIgM containing immunoglobulin composition obtained from step (c) it ispossible to lower the necessary irradiation time with UVC to achieve avirus inactivation of non enveloped viruses of more than 3 or 4 log₁₀.This results in a higher yield of native and active IgM during UVCtreatment.

Surprisingly these steps lead to a chemically and enzymaticallyunmodified IgM containing solution which has higher yields of native andactive IgM, having low anticomplementary activity and low proteolyticactivity and having high antibacterial and antiviral activity, with anoutstanding virus safety concerning enveloped and non enveloped viruses;a key feature for pharmaceuticals which are for intravenousadministration. Moreover, a treated IgM containing solution has improvedlong term stability being very stable in liquid solution for more than12 months at 2-8° C.

Medical Use

The antibody preparation of the present invention is suitable for use inmedicine and can be used in the treatment of immunological disorders andinfections, particularly IgM deficiency disorder and bacterialinfections. Human IgM-enriched polyvalent immunoglobulin preparation forintravenous administration contain higher antibody titres againstclinically relevant Gram-negative and Gram-positive bacteria as well ashigher antibody titres against endotoxins of Gram-negative bacteria andexotoxins of Gram-negative and Gram-positive bacteria compared withpolyvalent immunoglobulin G preparations.

In particular, the antibody preparations of the present invention aresuitable for intravenous administration to patients.

The invention also provides a method of treatment of a patientcomprising a step of administering the antibody preparation of thepresent invention to the patient. In particular, the patient can besuffering from an immunological disorder or a bacterial infection. In apreferred embodiment the antibody preparations are administeredintravenously.

The present invention will now be described further by way of exampleonly.

EXAMPLES

Assay Methods

Distribution of Molecular Size by HPLC for IgM Concentrate

The below method can be utilized to determine the % aggregates in anantibody preparation (as used in Example 8).

Test solution: Samples are injected undiluted at approx. 50 g/L with aninjection volume of 10 μl (approx. 500 μg protein load).

Reference solution: human immunoglobulin (e.g. Intratect, Biotest AG)

Standard solution: Bio-Rad gel filtration standard (Art.-No. 151-1901)

Column:

size: 1=30 mm, Ø=7.8 mm,

stationary phase: Tosoh Bioscience TSK-Gel G4000 SWXL, suitable forfractionation of globular proteins with relative molecular masses in therange 20 000 to 7×10⁶ Da.

Mobile phase: dissolve 4.873 g of disodium hydrogen phosphate dihydrate,1.741 g of sodium dihydrogen phosphate monohydrate, 11.688 g of sodiumchloride and 50 mg of sodium azide in 1 litre of water.

Flow rate: 0.5 ml/min

Detection: spectrophotometer at 280 nm. In the chromatogram obtainedwith the reference solution,

The chromatogram is integrated according to the following scheme and thepeaks are identified:

Polymer (>1200 kD), 10-13 min

IgM (1200-750 kD), 13-19 min

Dimer/IgA (750-350 kD), 19-20 min

IgG (350-100 kD), 20-26 min

Fragments (<100 kD), 26-40 min

Fragments (<100 kD), 26-40 min

Determination of Unspecific Complement Activation

Hemolysin pre-treated sheep erythrocytes are hemolysed by complement. Bycomplement-binding antibodies in the sample the hemolysis is suppressed.The amount of complement is determined, which is bound (inactivated) by1 mg immunoglobulin.

A certain amount of immunoglobulin (10 mg) is mixed with complement ofguinea pig and the free complement is titrated. The anti-complementaryactivity is expressed a used complement in respect to the usedcomplement of a reference solution. The hemolytic unit of complementactivity (CH₅₀) is the amount of complement leading to hemolysis of2.5×10⁸ optimally prepared erythrocytes of a total amount of 5×10⁸erythrocytes in optimal buffer conditions.

Optimally prepared erythrocytes (8 ml stabilized erythrocytes fromsheep, washed three times with gelatine-barbital-buffer, finally 1 mlerythrocyte sediment are suspended in 24 ml gelatine-barbital-buffer)are prepared by mixing 20 ml erythrocytes suspension with 20 mlhemolysine (adjusted to 2 MHE/ml—minimal hemolytic unit) and incubationfor 15 min at 37° C.

An equivalent of 10 mg immunoglobulin is diluted ingelatine-barbital-buffer (1 g gelatine in 1 L barbital buffer pH 7.3,5-fold barbital-buffer solution: 83 g sodium chloride, 10.192 g sodiumbarbital in 2 liters water, pH 7.3). To a final volume of 1 ml 200 μlcomplement 100 CH₅₀/ml are added. The test tubes are incubated undershaking for 1 h at 37° C. The samples are diluted and titrated againstoptimally prepared erythrocytes. After an incubation for 1 h at 37° C.the samples are centrifuged and the optical density is determined byusing a spectrophotometer at a wavelength of 541 nm.

Determination of Proteolytic Activity

Proteolytic activity can be assessed by mixing a chromogenic substrate(in particular those sensitive to at least one serine protease) and asample of the antibody preparation (usually diluted in buffer to meetthe linear range of the assay) at 37° C. and monitoring the absorptionkinetics using a spectrophotometer. The proteolytic activity of thesample is calculated from the initial absorption difference (ΔAbs/min)by using the equation C (U/L)=S×ΔAbs/min×F (C=proteolytic activity;S=conversion factor relating to specific adsorption change of thechromogenic substrate; and F=dilution factor). Use of the substrate isaccording to manufacturer's instructions.

The proteolytic activity can in particular be assessed via the followingsteps:

-   -   (a) 25 mg of the substrate S-2288 (Chromogenix) is dissolved in        7.2 ml of water-for-injection;    -   (b) a sample of the antibody preparation is diluted into buffer        (100 mM Tris.HCl pH 8.4, 106 mM NaCl) to meet the linear range        of the assay and temperature is adjusted to 37° C.;    -   (c) equal amounts (e.g. 200 μl) of the diluted antibody        preparation and the dissolved substrate are mixed;    -   (d) the absorption kinetics are measured at 405 nm for 1 to 3        minutes at 37° C. using a spectrophotometer;    -   (e) the proteolytic activity of the sample is calculated from        the initial absorption difference (ΔAbs/min) by using the        equation C (U/L)=313×ΔAbs/min×F (C=proteolytic activity,        F=dilution factor)

The limit of quantitation of this method is 8 U/l, and using a sample ofthe antibody preparation of the present invention proteolytic activityis undetectable. As such the level of the proteolytic activity in thefinal product of the present invention is below 8 U/l.

Example 1—Preparation of an IgM Enriched Preparation from Fraction I/III

180 kg Cohn Fraction originating from cold ethanol fractionation ofhuman plasma are suspended in 720 L 0.1 M sodium acetate buffer pH 5.05and mixed for 15-30 minutes after the suspension temperature is reached(22±4° C.).

The solution is treated by addition of a 19.8 kg octanoic acid (0.110 kgper kg paste I/III used) at room temperature and the protein solution isfurther mixed for 80 minutes, using a vibrating mixer (Vibromixer®, Size4, Graber+Pfenniger GmbH, Vibromixer adjusted to level 2-3). Theoctanoic acid is added slowly over 30 min

Approx. 3 kg tri-calcium phosphate (Ca₃(PO₄)₂) are added and the proteinsolution is further mixed for at least 15 min. The precipitate isremoved by clarifying filtration using a filter press. An additional 0.2μm filtration is carried out and the protein solution is subjected toultrafiltration with 10 kD membranes. The protein solution isdiafiltered against 0.04 M NaCl solution and afterwards adjusted to aprotein concentration of 40 g/L.

The protein solution is treated at pH 4.0±0.1 after dilution 1+1 withwater for injection. pH adjustment is carried out by using 1 M HCl andthe protein solution is incubated for 9 h at 37° C.±2° C. After the pH 4incubation the protein solution is adjusted to pH 5.8, using 1 M NaOH.The resulting protein solution is further purified by adding DEAESephadex in a batch mode (75 g DEAE Sephadex per kg protein). Theprotein solution is incubated under stirring for ≥60 min at roomtemperature. The DEAE Sephadex is removed by clarifying filtration. Theprotein solution is subjected to a 0.2 μm filtration.

The protein solution is filtered through a 0.1 μm filter and a Pall,Ultipor VF DV50, 20″ filter. The filtrate is further processed by UVClight treatment at 254 nm, using a flow-through UVivatec® process device(Bayer Technology Services/Sartorius Stedim) at a UVC dose of 240 J/m².The flow velocity through the UVC reactor is calculated using themanufactures instructions. The irradiated protein solution isconcentrated to a protein concentration of 50-70 g/l by ultrafiltrationand is subjected to diafiltration (10 kD membrane, using 0.32 M glycinebuffer pH 4.3). The final product is filtered through a 0.2 μm filterand is stored at 2 to 8° C.

Example 2—Investigation of Conditions in Octanoic Acid Treatment Step

For the octanoic acid treatment the following experimental ranges weretested, also in combination with each other using the method describedin Example 1 (results not shown).

-   -   Octanoic acid amount: 0.09 kg/kg to 0.13 kg/kg (Amount octanoic        acid per kg used fraction I/III) (120 to 180 mM octanoic acid)    -   pH of the octanoic acid treatment between pH 4.8 and 5.3    -   Temperature range of the reaction: 14° C. to 30° C.    -   Incubation time: 40 to 110 min

All conditions tested lead to intermediates being easy to clarify forfurther processing and with a extensive reduction of proteolyticactivity from several thousand U/L in suspended Cohn fraction I/III).These intermediates result in a final product with a proteolyticactivity below 8 U/l (calculated as described below in Example 6) whichis the limit of quantitation.

Example 3—Virus Reduction Through Use of Vibromixer—Determination ofVirus Removal Factors for the Octanoic Acid Treatment with and withoutUse of a Vibromixer

250 ml of suspended faction I/III were homogenised for 30 min at pH 5.05and 22° C. The suspension was spiked with 2.6 ml of the virus stocksolution. Octanoic acid was added (110 g/kg) and homogenised for 60 minusing a vibromixer. In a parallel experiment the same mixture washomogenised with standard stirring. After 60 min tri-calcium phosphate(0.15 g/kg octanoic acid) was added and the suspension stirred for 15min. The suspension was cleared by depth filtration using a filter disc.The filter disc was pre-rinsed with 70-80 ml of buffer. Afterfiltration, the filter was rinsed with 80 ml of buffer. Filtrate andwash were pooled and a sample was drawn for virus titration.

Virus titres from samples taken prior to addition of octanoic acid andafter filtration were determined on appropriate indicator cells forSV40, Reo and PPV (CV-1, CCL.7.1 and PK13). Finally, the removal factorwas calculated in compliance with the current guidelines for virusvalidation studies.

In virus validations studies, non-enveloped viruses such as SV40 and Reowere effectively removed in the order of more than 4 log₁₀ and more than5 log₁₀, respectively. Moreover, PPV was removed by more than 3 log₁₀.These values are more than 10 times and up to 1000 times higher thanwith the same octanoic acid treatment under standard stirring conditionswithout vibromixing.

TABLE 1 Comparison of the virus reduction factors (log₁₀) for theoctanoic acid treatment with and without the use of a vibromixer.Octanoic acid Octanoic acid reaction reaction standard stirring withvibromixing [log₁₀ reduction] [log₁₀ reduction] PPV 2.15 ± 0.32 3.39 ±0.36 REO 2.34 ± 0.38 5.46 ± 0.28 SV40 2.05 ± 0.4  4.71 ± 0.34

Example 4—Evaluation of UVC Treatment

The optimal range for the dosage of UVC radiation has been evaluated.There is a balance between the minimal necessary dosage to achieve atleast 4 log₁₀ inactivation for non enveloped viruses and the maximumtolerable dosage to avoid denaturation of the IgM molecules leading toan impaired Fab function to bind antigens and impaired Fc functioninfluencing complement activation. In the range of 200 to 400 J/m² onecould observe only a slight increase of immunoglobulin aggregates and nosignificant impact on fragment content.

For the experiments the optical density (OD) of the original proteinsolution is used to calculate the flow rate in the UVivatec lab systemwith the vendor provided Excel-Sheet from BTS (customer MasterCalculation Sheet UVivatec Lab II Version 3.0). The flow rate iscalculated by taking into account the lamp performance, the set point ofthe UV signal lamp sensor and the desired UVC irradiation dose.

IgM containing solution with a protein content of about 55 g/l (Batch86GB005BE07) was pumped at a flow rate of 5.8 l/h through the UVivatecsystem in order to achieve a dose of 200 J/m² for a single flow-through.A dose of 300 J/m² was achieved by pumping the protein solution at aflow rate of 3.9 L/m² through the system. 400 J/m² were achieved bypumping the protein solution at a flow rate of 2.9 L/m² through thesystem.

TABLE 2 Analytical results for the activity and titre determinationsbefore and after UVC treatment in the concentrated final product IgMproduct IgM product IgM product IgM product no UVC- after UVC after UVCafter UVC irradiation 200 J/m² 300 J/m² 400 J/m² Protein content g/l56.3 56.2 57.6 54.4 IgG content % 56.1 55.5 55.7 54.9 (nephelometry) IgAcontent % 20.1 20.6 20.5 20.7 (nephelometry) IgM content % 23.7 23.923.7 24.4 (nephelometry) HPSEC aggregates >1200 kD area % 1.9 2.6 3.34.0 fragments <100 kD area % 0.66 0.73 0.76 0.79 ACA CH50/mg 0.68 0.480.46 0.46 protein PA U/l <8 <8 <8 <8

No significant difference could be observed for immunoglobulin content,proteolytic activity or ACA in the range of 200 to 400 J/m². Thepreferred range for the dosage was set between 200 and 300 J/m² because200 J/m² are well enough to inactivate non enveloped viruses and at 300J/m² no significant impact could be seen on aggregate formation andantibody titres. The preferred dosage is 225 J/m2

Diluted IgM containing solution with a protein content of 8 to 12 g/l(Batch 86BB059BE07) was pumped at a flow rate of 5.8 l/h through theUVivatech system in order to achieve a doses between 200 and 300 J/m²for a single flow-through.

TABLE 3 Analytical results for IgM solutions before and after UVCirradiation at different UVC doses Batch fraction I/III before UVC: UVC:UVC: UVC: 86BB059BE07 UVC 200 J/m² 225 J/m² 250 J/m² 300 J/m² Proteing/l 11.34 10.56 10.65 10.69 10.56 IgG content % 59.2 59.1 58.5 58.6 57.1IgA content % 19.6 19.6 20.2 20.1 20.3 IgM content % 21.1 21.3 21.2 21.422.6 HSEC aggregates >1200 kD % 0.20 0.39 0.54 0.3 0.47 fragments <100kD % 0.47 0.46 0.25 0.26 0.47 PA U/l <8 <8 n.t. n.t. n.t. PKA U/ml 3 3 33 3 ACA CH50/ 0.1 0.08 0.1 0.1 0.18 mg protein Anti-E. coli O1:K1 - U/mg24.7 20.5 18.9 19.5 20.2 IgG Anti-E. coli O1:K1 - U/mg 9.4 9.5 9.5 9.18.9 IgA Anti-E. coli O1:K1 - U/mg 14.1 13.0 15.1 13.9 13.4 IgMAnti-Candida U/mg 15.6 16.8 17.9 17.3 17.0 albicans - IgG Anti-CandidaU/mg 11.3 11.6 10.5 10.3 10.4 albicans - IgA Anti-Candida U/mg 13.8 13.313.7 13.9 13.1 albicans - IgM Anti-Enterococcus U/mg 13.0 15.5 13.5 14.815.0 faecalis - IgG Anti-Enterococcus U/mg 11.3 10.5 10.1 9.7 9.6faecalis - IgA Anti-Enterococcus U/mg 17.2 14.1 16.7 14.0 13.9faecalis - IgM Anti-Pneumococcus U/mg 23.2 24.1 24.7 24.0 25.7 Saccharid-IgG Anti-Pneumococcus U/mg 13.3 12.1 18.0 16.5 14.8 Saccharid -IgAAnti-Pneumococcus U/mg 17.5 15.1 18.0 16.4 16.6 Saccharid -IgM

The distribution between the immunoglobulin classes remains unaffectedby the UV irradiation within this dosage range procedure. The molecularweight distribution pattern analyzed by HPSEC is also not changed. Thelevel of purity analyzed by CZE remains unchanged. Proteolytic activity(PA), prekallikrein activator (PKA) and anti-complementary activity(ACA) are unchanged. Also the anti bacterial activity measured by anElisa method are not significantly altered for all immunoglobulinclasses.

The aliquots—irradiated with increasing UV intensities—were furtherprocessed until final product and subjected to the same panel ofanalytical tests. There was also no significant difference observable inthe final products. All tested antibody titres are always in the rangeof 100±10% of the control preparation not UVC treated.

Example 5—Overall Virus Reduction Through Use of Vibromixer/pH4Treatment and UVC Treatment—Determination of Virus Removal Factors

The validation of virus removal/inactivation of the three steps octanoicacid treatment with vibromixing, pH4 treatment and UVC treatment (215J/m²) was performed using the following model viruses: Bovine ViralDiarrhea Virus (BVDV) as model virus for Hepatitis C Virus, PseudorabiesVirus (PRV) as model virus for Human Herpes Viruses, HumanImmunodeficiency virus (HIV-1), Equine Arteritis Virus (EAV) as modelvirus for corona viruses, Sindbis Virus (SinV) as model virus for Flaviviruses, Murine Encephalomyelitis Virus (MEV) as model virus forHepatitis A Virus, Reovirus (Reo) as model virus for other non envelopedviruses, Porcine Parvovirus (PPV) as model virus for human ParvovirusB19.

The results of these studies with the three steps octanoic acidtreatment, pH4 treatment and UVC treatment are listed in the followingTable 2.

TABLE 4 Total virus reduction by the IgM production process Model virusBVDV PRV HIV-1 EAV SinV MEV Reo PPV Totalreduction >12.5 >10.1 >12.7 >8.4 ^(a) >13.7 ^(a) 9.2 >11.0 >8.4 (log₁₀)^(a) Reduction factor without data for validation of the UVC irradiationstep

The optional nano filtration with filters with a nominal pore size ofabout 50 nm adds additional safety by increasing the total reduction upto more than 17 log 10 depending on the size of the virus. E.g >17.5log₁₀ are then reached for HIV-1 whereas PPV was not further removed bynano filtration.

Therefore the purification procedure according to the invention leads toan outstanding virus safe IgM preparation with up to now for such an IgMcontaining preparation unreached virus inactivation/reduction rates ofmore than 8 log₁₀. This is especially important for the non envelopedviruses like MEV, Reo and PPV which are generally more resistant againstvirus inactivation and removal procedures due to their small size andthe lack of a lipid envelope.

Example 6: Determination of Residual Proteolytic Activity for theOctanoic Acid Treatment with and without Use of a Vibromixer

The octanoic acid treatment was performed like in example 1 and in aparallel experiment without a vibromixer but with vigorous standardstirring with a blade stirrer. The proteolytic activity in samples afteroctanoic acid/tricalcium phosphate treatment and ultra-/diafiltrationwere determined using the chromogenic substrate S-2288 (Chromogenix),following the manufacturers instructions.

25 mg of the substrate S-2288 (Chromogenix) are dissolved in 7.2 mlwater-for-injection. Samples are diluted into buffer (100 mM Tris/HCl pH8.4, 106 mM NaCl) to meet the linear range of the assay, e.g. 200 μlbuffer are mixed with 200 μl sample (mixing and temperature adjustmentto 37° C.) and 200 μl chromogenic substrate solution. The absorptionkinetics are measured at 405 nm (1-3 min) at 37° C., using aspectrophotometer. The proteolytic activity of the sample is calculatedfrom the initial absorption difference (ΔAbs/min) by using the equationC (U/L)=313*ΔAbs/min*F (C=proteolytic activity, F=dilution factor)

TABLE 5 Reduction of proteolytic activity by octanoic acid treatmentOctanoic acid Octanoic acid treatment without treatment with vibromixingvibromixing Starting material (U/l) 5630 5630 Mean residual proteolytic42 <8 (LOD) activity after octanoic acid treatment (U/L)

The filtrate after octanoic acid treatment was limpid when vibromixingwas employed. In the comparison experiment the filtrate after octanoicacid treatment with blade stirrer was very opaque and difficult tofiltrate.

Example 7: Anti-Bacterial Titres in an IgM Preparation According to theInvention

For comparison with the only commercially available intravenoustolerable IgM containing preparation Pentaglobin, the anti-bacterialactivities were analyzed in three batches of this well established drugand compared to a preparation according to the invention. Thedetermination of antibodies of the IgA or IgM class in the IgMpreparation versus antibacterial or antifungal antigens was carried outby ELISA. Microtitre plates were coated with a corresponding antigen andincubated with a standard or the IgM preparation. Antibodies bound tothe antigen were detected with an anti-human-IgA or anti-human-IgMconjugate. The detection was carried out by using an enzyme substrate.The resulting colour change is corresponding to the amount of antibodiespresent in IgM preparation.

TABLE 6 Comparison of anti bacterial binding activity of IgM in anpreparation according to the invention and commercially availablePentaglobin IgM Pentaglobin preparation commercial parameter unitinvention mean product mean IgM antibodies against U/mg IgM 72 21Pneumococcus saccharide IgM antibodies against U/mg IgM 62 39Escherichia coli IgM antibodies against U/mg IgM 69 27 Enterococcusfaecalis IgM antibodies against U/mg IgM 61 41 Candida albicans IgMantibodies against U/mg IgM 71 6 Chlamydia

TABLE 7 Comparison of anti bacterial binding activity of IgA in anpreparation according to the invention and commercially availablePentaglobin IgM Pentaglobin preparation commercial parameter unitinvention mean product mean IgA antibodies against U/mg IgA 86 25Pneumococcus saccharide IgA antibodies against U/mg IgA 83 26Escherichia coli IgA antibodies against U/mg IgA 93 21 Enterococcusfaecalis IgA antibodies against U/mg IgA 65 38 Chlamydia IgA antibodiesagainst U/mg IgA 59 24 Helicobacter pylori

The IgM and IgA mediated activities in the new preparation weretypically at least 1.5 times as high as in Pentaglobin which can beexplained by the fact that IgM and IgA in Pentaglobin is chemicallymodified with β-Propiolactone This step is replaced by the more gentleprocedures according to this invention.

Overall these data demonstrate that the binding region of the IgMmolecules in the final preparation is functionally full active.

Example 8 Storage Stability Studies with Liquid IgM Product

Product according Example 1 without UVC treatment was stored in 10 or100 ml glass vials (filling volume 5 ml or 50 ml) at 2-8° C. andanalyzed for all parameter according to specification. The results areshown in Table 8. Parameter which are relevant to show stability are theaggregate and fragment content measured with high performance sizeexclusion chromatography (HPSEC), proteolytic activity (PA) andanticomplementary activity (ACA). These parameter are critical forintravenous tolerability and likely to change during long term storage.At 2-8° C. there was no significant change of these parameters. Even atstorage at room temperature (23-27° C.) these values remained withinspecification, although there is a slight increase of fragments after 24months at room temperature. Other parameter like coloration,opalescence, pH value were also determined and stayed unchanged over thewhole study period. IgM and IgA titres against various bacteria remainstable over 2 years at 2-8° C.

Product according example 1 with UVC treatment was also stored in 10 or100 ml glass vials (filling volume 5 ml or 50 ml) at 2-8° C. and roomtemperature and analyzed for all parameter according to specification.The results are shown in Table 9. In this ongoing stability study thecurrently available 12 month date show the same stability profile of theproduct as without UVC treatment which allows the extrapolation to a 24month stability.

TABLE 8 Stability of the batch A586067 tested at 2-8° C. lying positionFilling size: 5 ml Requirement Storage in months 2-8° C. 23-27° C.Parameters tested (Tolerance) 0 3 6 9 12 18 24 24 protein (g/1) 45-5550.3 51.4 50.3 50.4 50.5 49.6 50.8 49.8 HPSEC % aggregates >1200 kD ≤50.9 0.6 0.5 0.8 0.6 1.0 1.3 1.7 % fragments <100 kD ≤5 0.2 0.6 1.1 0.71.6 0.9 1.2 4.1 proteolytic activity (U/l)  <8 <8 <8 <8 n.t. <8 n.t. <8<8 immunoglobulin content  >95% 96.7 99.0 100 n.t. 99.5 n.t. 98.4 97.5(%) IgM content ≥20% 21.6 22.1 22.1 n.t. 22.3 n.t. 20.9 20.5anticomplementary   ≤1.0 0.48 0.56 0.48 0.66 0.70 0.64 0.54 0.38activity (CH50/mg protein) n.t. = not tested

TABLE 9 Stability of the batch A586057 tested at 2-8° C. lying positionFilling size: 50 ml Requirement Storage in months 2-8° C. 23-27° C.Parameters tested (Tolerance) 0 3 6 9 12 18 24 24 protein (g/l) 45-5550.2 50.8 49.7 50.4 50.3 49.4 50.3 49.7 HPSEC % aggregates >1200 kD ≤50.9 0.5 0.4 0.8 0.6 1.0 1.3 1.5 % fragments <100 kD ≤5 0.3 0.6 1.0 0.91.4 1.2 1.2 4.2 proteolytic activity (U/l)  <8 <8 <8 <8 n.t. <8 n.t. <8<8 immunoglobulin content  >95% 98.6 98.9 100 n.t. 99.5 n.t. 98.5 98.0(%) IgM content ≥20% 21.3 22.3 24.5 n.t. 22.0 n.t. 20.9 20.1anticomplementary   ≤1.0 0.48 0.82 0.52 0.64 0.68 0.48 0.60 0.40activity (CH50/mg protein)

Example 9 In Vitro Unspecific Complement Activation with the IgM ProductExample 9A—Determination of C5a Levels

Analysis of the potential of the IgM preparation to activate complementin vitro unspecifically was performed using factor C5a as a marker foractivation of the terminal complement pathway. For this purpose humanserum was incubated with immunoglobulin products or buffer for 120 min.Samples were taken after 0, 5, 15, 60 and 120 minutes of incubation. Inorder to demonstrate appropriate function of the in vitro systemcomplete inhibition as well as full activation of the complement systemwas shown. The complement factor concentration was measured via opticaldensity determinations with a photometer using a commercial availableenzyme linked immunosorbent assay (ELISA) kit (Quidel MicroVue C5a PlusEIA Kit; A025).

Human serum (Quidel NHS; A113) was thawed quickly at 37° C. andimmediately put on ice. Every single sample consisted of a reactionbatch containing serum (100 μl). Additives were first pipetted followedby the addition of human serum to start the reaction in every reactionbatch.

Human serum without any additives served as a blank and showed baselinecomplement activation due to the experimental setup. Addition of heataggregated IgG (HAAG Quidel; A114; 1.3 μl) served as a strong activatorof human serum complement to demonstrate the responsiveness of the invitro system. EDTA (final concentration 10 mM) was added to human serumin order to completely inhibit complement activation over the entirereaction time and experimental treatment. IgM preparation, Pentaglobin(according to EP0013901) and an IgM preparation according to EP0413187were adjusted to an IgM-concentration of 1.72 mg/ml in each reaction. Asnegative controls the respective volume of formulation buffer was used.

All reactions were stopped after incubation for 0, 5, 15, 60 and 120minutes at 37° C. under constant agitation by addition of stabilizingsolution (Quidel sample stabilizer A9576; 140 μl). Subsequent sampledilution and ELISA analysis was performed following the manufacturer'sprotocol. The experiment was performed in two independent replicates andmean values were calculated. The results are shown in Table 10 and FIG.2.

Addition of activator (heat aggregated IgG) lead to a strong increase ofC5a within 15 minutes indicating a sensitive response of the in vitrosystem to detect complement activation. The addition of EDTA asinhibitor resulted in unchanged values over the entire incubation timeshowing that complement activation is specific and not an artefact dueto sample handling or preparation. Incubation of human serum at 37° C.and exposure to artificial surfaces induced a slight complementactivation documented as blank values.

The IgM reference preparation according to EP0413187 resulted incomplement activation up to more than 1000 ng/ml after 60 Minutes (table10). The commercially available chemically modified referencepreparation Pentaglobin (EP0013901) still showed half of the complementactivating potential compared to the EP0413187 product.

The concentration of C5a in serum treated with the IgM preparationaccording to this invention is comparable to the C5a concentrationmeasured in serum without additives (blank) or in serum treated with theformulation buffers (300 mM Glycin, pH 4.3 or 0.45% NaCl/2.5% Glucose,pH 6.8). Thus the immunoglobulins in the IgM preparation according tothis invention substantially do not unspecifically activate complementin human serum in the in vitro test system.

TABLE 10 Mean C5a concentration detected in human serum treated with IgMcontaining immunoglobulins Time [min] 0 5 15 60 120 IgM preparation(invention) 23.8 42.0 110.5 162.0 150.8 C5a [ng/ml] Pentaglobin(EP0013901) 46.4 55.4 329.2 460.9 653.5 C5a [ng/ml] EP0413187 product21.1 149.5 423.2 1029.4 1084.2 C5a [ng/ml] Controls Blank 22.3 30.7 66.2149.5 168.1 C5a [ng/ml] Activator (IgG polymers) 19.4 897.6 3409.24536.1 4829.6 C5a [ng/ml] Inhibitor EDTA 25.9 22.3 25.5 23.8 27.5 C5a[ng/ml] Formulation buffer IgM 19.8 35.5 101.2 112.8 173.4 C5a [ng/ml]Formulation buffer 26.2 33.1 56.7 82.6 187.2 Pentaglobin C5a [ng/ml]

Example 9B Determination of C3a Levels

Analysis of the potential of the IgM preparation to activate complementin vitro unspecifically was performed using factor C3a as a marker foractivation of the complement pathway. For this purpose human serum wasincubated with immunoglobulin products or buffer for 120 min. Sampleswere taken after 0, 5, 15, 60 and 120 minutes of incubation. In order todemonstrate appropriate function of the in vitro system completeinhibition as well as full activation of the complement system wasshown. The complement factor concentration was measured via opticaldensity determinations with a photometer using a commercial availableenzyme linked immunosorbent assay (ELISA) kit (Quidel MicroVue C3a PlusEIA Kit; A032).

Human serum (Quidel NHS; A113) was thawed quickly at 37° C. andimmediately put on ice. Every single sample consisted of a reactionbatch containing serum (100 μl). Additives were first pipetted followedby the addition of human serum to start the reaction in every reactionbatch.

Human serum without any additives served as a blank and showed baselinecomplement activation due to the experimental setup. Addition of cobravenom factor (CVF Quidel; A600; 20 U/ml) served as a strong activator ofhuman serum complement to demonstrate the responsiveness of the in vitrosystem. EDTA (final concentration 10 mM) was added to human serum inorder to completely inhibit complement activation over the entirereaction time and experimental treatment. IgM preparation andPentaglobin (according to EP0013901) were adjusted to anIgM-concentration of 1.72 mg/ml in each reaction. As negative controlsthe respective volume of formulation buffer was used.

All reactions were stopped after incubation for 0, 5, 15, 60 and 120minutes at 37° C. under constant agitation by addition of stabilizingsolution (Quidel sample stabilizer A9576; 140 μl). Subsequent sampledilution and ELISA analysis was performed following the manufacturersprotocol. The experiment was performed in two independent replicates andmean values were calculated. The results are shown in Table 11 and FIG.3.

Addition of activator (CVF) lead to a strong increase of C3a within 15minutes indicating a sensitive response of the in vitro system to detectcomplement activation. The addition of EDTA as inhibitor resulted inunchanged values over the entire incubation time showing that complementactivation is not an artefact due to sample handling or preparation.Incubation of human serum at 37° C. and exposure to artificial surfacesinduced a slight complement activation documented as blank values.

The commercially available chemically modified reference preparationPentaglobin (EP0013901) showed a three times higher C3 activatingpotential compared to the blank.

The concentration of C3a in serum treated with the IgM preparationaccording to this invention is comparable to the C3a concentrationmeasured in serum without additives (blank) or in serum treated with theformulation buffers (300 mM Glycin, pH 4.3 or 0.45% NaCl/2.5% Glucose,pH 6.8). Thus the immunoglobulines in the IgM preparation according tothis invention substantially do not unspecifically activate complementin remarkable amounts in human serum in the in vitro test system.

TABLE 11 Mean C3a concentration detected in human serum treated with IgMcontaining immunoglobulins Time [min] 0 5 15 60 120 IgM preparation1458.3 2484.1 3972.1 5280.7 5703.1 (invention) C3a [ng/ml] Pentaglobin(EP0013901) 1371.9 3069.4 7585.9 10225.4 11769.5 C3a [ng/ml] ControlsBlank 1301.1 1742.6 2468.7 3361 4117.4 C3a [ng/ml] Activator (CVF)1194.3 6077.1 12796.8 27679.1 27284.5 C3a [ng/ml] Inhibitor EDTA 1140.21098.0 1025.7 964.2 1004.8 C3a [ng/ml] Formulation buffer IgM 1060.32262.3 2907.3 3480.7 4435.4 C3a [ng/ml] Formulation buffer 1070.9 1965.83548.0 4008.1 5251.9 Pentaglobin C3a [ng/ml]

Example 10 In Vivo Experiments with IgM Product

To confirm safety and tolerability the effects of the IgM preparation onarterial blood pressure following repeated intravenous infusions over 5days were studied in 8 conscious cynomolgus monkeys. A dose of 190mg/IgM/kg/day of the IgM preparation prepared according to the methodsdescribed herein was administered. Pentaglobin, the commerciallyavailable intravenous tolerable IgM containing preparation wasadministered to some monkeys as a comparison substance. Pentaglobin wasadministered in such a way that the same IgM dose was administered.Blood pressure was determined following injection to determine whetheradministration was associated with an intolerable level of unspecificcomplement activation. A control dose of 0.9% NaCl was administered tothe animals several hours prior to the administration of theimmunoglobulin preparations. Blood pressure was determined by insertinga pressure catheter into the lower abdominal aorta via the right femoralartery. Results were transmitted by telemetry.

The administration of the IgM preparation (15 ml/kg/day) had only minoreffects on arterial blood pressure (mean, systolic and diastolic). Thedifferences up to 4 hours after every infusion compared to pretestvalues did not exceed 4 mmHg. These differences can be considered notbiologically relevant.

TABLE 12a C3a levels [ng/ml] after the administration of the IgMpreparation Control (0.9% NaCl, pH 4.5) Administration of IgMpreparation C3a [ng/ml] C3a [ng/ml] Mean 229 240 SD 83 37 N 8 8

TABLE 12b C3a levels [ng/ml] after the administration of the referencepreparation Pentaglobin Control (0.9% NaCl, pH 6.8) Administration ofIgM preparation C3a [ng/ml] C3a [ng/ml] Mean 204 263 SD 20 61 N 4 4

C3a levels were determined in plasma samples taken after injection as amarker for unspecific activation of the complement pathway. C3a levels[ng/ml] were only slightly increased by the administration of the IgMpreparation (15 mL/kgBW) and were even lower than with the commerciallyavailable reference preparation Pentaglobin at equal amounts of IgM.Blood samplings were performed approximately 6 hours after treatment.

No substantial toxicological findings could be attributed to the IgMpreparation, and there were no relevant alterations that have not beenobserved with Pentaglobin. As the safety of Pentaglobin is wellestablished in the clinical practice of many years it is reasonable toconclude that these alterations do not have any clinical relevance.

The good tolerability and safety of the IgM preparation was alsoverified in a human Phase I study in 24 healthy male and femalevolunteers. Systolic blood pressure in the first 4 hours afteradministration in the mean decreased only about 9% (11.9 mmHg) afterinfusion of 91 to 274 mg of the IgM preparation per kg BW/d at 0.5ml/min.

This was in the same range like the placebo 0.9% NaCl-solution (9.4%,11.7 mmHg).

No serious adverse events were recorded and all non-serious adverseevents were self limiting. Further, there was no evidence for thetransmission of an infectious agent, as shown by PCT determinations.

It is noted that the usefulness of efficacy studies in animal models ofrelevant diseases is limited due to the immunogenicity and preformedGal-antibodies in IgM preparations obtained from human plasma. However,given the prior art knowledge regarding the use of Pentaglobin in thetreatment of disease and the anti-bacterial antibody titres of the IgMpreparation prepared by the method of the present invention (asdemonstrated in Example 7) it can be concluded that the IgM preparationhave clinical efficacy.

Example 11 Functional Integrity of the Fc Part of the AntibodyPreparation

Functional integrity of the Fc part of the antibodies in the antibodypreparation prepared in accordance with the method described herein wasanalysed using the current Ph. Eur. method (2.7.9 Test for Fc Functionof Immunoglobulins Eur. Ph. Edition current April 2011) according to theEuropean Guidelines ICH S6 (CPMP/ICH/302/95) (Note for Guidance onpreclinical safety evaluation of biotechnology-derived pharmaceuticals)for IgG preparations. The European Pharmacopoeia's monograph forimmunoglobulins (01/2005:20709) proposes a Rubella antigen-based testfor Fc function of immunoglobulins.

In particular, tanned group O human red blood cells were loaded withrubella virus antigen. Specific volumes of the antibody preparationswere incubated with antigen coated blood cells. The complement-initiatedlysis of the blood cells was started by adding guinea pig complement.The kinetics of subsequent haemolysis was measured via time-dependentchanges of absorbance at 541 nm. The evaluation was carried out usingthe maximal change of absorbance per time. Human ImmunoglobulinBiological Reference Preparation; BRP Batch no. 3 was used as thecomparison.

The activity of the Fc part of the antibody molecule was determined in 7batches of the IgM containing antibody preparation and was in allbatches between 96.5 and 103.3% compared to the biological referencepreparation (BRP), therefore proving the functionality of the IgMcontaining antibody preparation.

We claim:
 1. A method of treatment of a patient suffering from a viralinfection comprising administering an antibody preparation to saidpatient wherein the antibody preparation comprises immunoglobulins IgG,IgA and IgM, wherein at least 15% by weight of the total immunoglobulinsare IgM, at least 5% by weight of the total immunoglobulins are IgA andat least 40% by weight of the total immunoglobulins are IgG, wherein theantibody preparation is virus safe with respect to enveloped andnon-enveloped virus, has an anticomplementary activity of ≤1 CH 50/mgprotein, is stable in liquid form for at least 2 years when stored at 2to 8° C., wherein the antibody preparation comprises a proteinconcentration of 40 g/L to 80 g/L, and an immunoglobulin content ofgreater than 95% of the total protein content.
 2. A method of treatmentaccording to claim 1 wherein the antibody preparation has a proteolyticactivity of less than 8 U/L.
 3. A method of treatment according to claim1 wherein the antibody preparation is administered intravenously.
 4. Amethod of treatment according to claim 1 wherein the immunoglobulinshave not been modified by contacting with β-propiolactone.
 5. A methodof treatment according to claim 1 wherein the antibody preparationcomprises less than 2% aggregates of 1200 kDa or above of the totalimmunoglobulin content as determined by high performance size exclusionchromatography.
 6. A method of treatment according to claim 1 whereinthe anticomplementary activity is less than 0.75 CH50/mg protein.
 7. Amethod of treatment according to claim 1 wherein at least 90% of theantibodies in the antibody preparation are biologically active.
 8. Amethod of treatment according to claim 1 wherein, in an in vitro Rubellaantigen-based assay suitable to determine Fc function, the activity ofthe Fc part of the antibodies of the antibody preparation is the same asthat of a biological reference preparation ±10%.
 9. A method oftreatment according to claim 1 wherein the antibody preparation has beenprepared in the absence of a step of heat treatment at a temperature 40°C. or above for more than 10 minutes.
 10. A method of treatmentaccording to claim 1 wherein the antibody preparation comprises a buffercomprising glycine and a pH in the range of pH 4 to 5.5.
 11. A method oftreatment according to claim 1 wherein the antibody preparation has beenprepared from human plasma.
 12. A method of treatment according to claim11 wherein the antibody preparation has been prepared from human plasmaby a process comprising the steps of: (a) preparing from the humanplasma a plasma fraction as a solution containing immunoglobulins; (b)mixing a C7 to C9 carboxylic acid with the solution and treating themixed solution with a vibrating agitator to precipitate contaminatingproteins; (c) separating the precipitated proteins from the solution toyield an IgM-containing immunoglobulin composition; (d) incubating theIgM-containing immunoglobulin composition at between pH3.5 and pH 4.5 toform an incubated solution; (e) irradiating the incubated solution withUVC to form a UVC-irradiated solution; and (f) filtering theUVC-irradiated solution under sterile conditions to form the antibodypreparation.
 13. A method of treatment according to claim 12 wherein thecarboxylic acid is octanoic acid.
 14. A method of treatment according toclaim 12 wherein the process further comprised subjecting the incubatedsolution obtained from step (d) to nanofiltration prior to saidirradiating step (e).
 15. A method of treatment of a patient sufferingfrom a viral infection comprising administering an antibody preparationto said patient wherein the antibody preparation comprises at least 15%IgM, more than 5% IgA and more than 40% IgG as percentages of the totalamount of antibodies, and comprises less than 1.5% aggregates of 1200KDa or above of the total immunoglobulin content as determined by highperformance size exclusion chromatography.
 16. A method of treatment ofa patient suffering from a viral infection comprising administering anantibody preparation intravenously to said patient wherein the antibodypreparation comprises IgG, IgA and at least 5% IgM antibodies by weightof the total amount of antibodies; wherein the preparation is preparedfrom human plasma; wherein the antibody preparation is prepared by aprocess which is capable of a more than 3 log₁₀ removal of non-envelopedviruses; wherein the antibody preparation has specific complementactivating activity; and wherein, in an in vitro assay with human serumsuitable to determine the ability of the antibody preparation toactivate complement unspecifically, the antibody preparation generates:(i) substantially no C5a, such that the antibody preparation, adjustedto an IgM concentration of 1.72 mg/ml, generates less than 200 ng/ml C5aafter 60 minutes of the assay and/or (ii) substantially no C3a, suchthat the antibody preparation, adjusted to an IgM concentration of 1.72mg/ml, generates less than 6000 ng/ml C3a after 60 minutes of the assay.17. A method of treatment according to claim 16 wherein the antibodypreparation comprises one or more of the following: (i) an amount of IgAthat is more than 5% by weight of the total amount of antibodies; (ii)an amount of IgG that is more than 40% by weight of the total amount ofantibodies; and (iii) an amount of IgM that is at least 15% by weight ofthe total amount of antibodies.
 18. A method of treatment according toclaim 16 wherein the anti-complementary activity of the preparation isless than 1.0 CH50/mg protein.
 19. A method of treatment according toclaim 16 wherein in the in vitro serum assay, the antibody preparationwith human serum generates the same amount of C5a and/or C3a as humanserum alone ±70%.
 20. A method of treatment according to claim 16wherein: (1) said in vitro serum assay to determine that the antibodypreparation generates substantially no C5a comprises the steps of:(a)(i) adding an amount of the antibody preparation to 100 μl humanserum to create a reaction mixture containing 1.72 mg/ml IgM andincubating the reaction mixture for 60 minutes at 37° C. with constantagitation; (b)(i) preparing a set of dilutions of the reaction mixturesuitable for an ELISA; (c)(i) performing a sandwich ELISA on the set ofdilutions of the reaction mixture utilizing a primary and a secondaryantibody to C5a and a chromogenic substance, wherein the secondaryantibody is conjugated to an enzyme and the chromogenic substance is thesubstrate of the enzyme; and (d)(i) determining the amount of C5a in thereaction mixture based on a colour change obtained as a result ofcontacting the chromogenic substance with the enzyme bound to C5a viathe secondary antibody, or (2) said in vitro serum assay to determinethat the antibody preparation generates substantially no C3a comprisesthe steps of: (a)(ii) adding an amount of the antibody preparation to100 μl human serum to create a reaction mixture containing 1.72 mg/mlIgM and incubating the reaction mixture for 60 minutes at 37° C. withconstant agitation; (b)(ii) preparing a set of dilutions of the reactionmixture suitable for an ELISA; (c)(ii) performing a sandwich ELISA onthe set of dilutions of the reaction mixture utilizing a primary and asecondary antibody to C3a and a chromogenic substance, wherein thesecondary antibody is conjugated to an enzyme and the chromogenicsubstance is the substrate of the enzyme; and (d)(ii) determining theamount of C3a in the reaction mixture based on a colour change obtainedas a result of contacting the chromogenic substance with the enzymebound to C3a via the secondary antibody.